8 research outputs found
Novel methods for high level ex vivo expansion of hematopoietic stem cells
The development of strategies to extensively expand HSCs ex vivo could
greatly improve the utility of hematopoietic stem cell (HSC) - based therapies. In
addition to potential clinical applications, such an advance would provide an
invaluable tool for studying the mechanisms underlying HSC self-renewal.
Engineered overexpression of the homeobox transcription factor HOXB4
has emerged as a powerful stimulator of hematopoietic stem cell (HSC)
expansion ex vivo (>40-fold net increase in 2 weeks). More recent studies of the
properties of natural and engineered NUP98-HOX fusion genes, initially of
interest to us for their role in human AML, suggested these molecules might have
similar effects on HSCs.
To examine whether specific NUP98 and HOX fusion genes stimulate
murine HSC expansion in short term liquid cultures, 3x10ⶠmarrow cells from
mice given 5-fluorouracil 4 days previously were prestimulated with IL-3, IL-6 and
SF, retrovirally transduced with MSCV-IRES-GFP retroviral vectors also
encoding NUP98-HOXB4, NUP98-HOXA10, or HOXB4 (only) or nothing as
controls and then cultured for another 6 days with the same growth factors.
Limiting dilution assays were used to determine the frequency and hence number
of Competitive long-term (>4months) lympho-myeloid Repopulating Units (CRU)
present before and after culture. The results of these experiments showed that
the CRU content of the cultures of NUP98-HOXB4-, and NUP98-HOXA10-
transduced cells increased 290-fold and >2000-fold, respectively, i.e. ~4 and
>25x the effect obtained with HOXB4 and >10⎠and >10â”x the yield of CRU in the
control cultures. Similar results were obtained in cultures of NUP98-HOXA10-
transduced cells that were initiated with limiting numbers of CRUs (1-2),
demonstrating that the cells targeted were not a rare subset of HSCs. Additional
studies of the same design showed that the effect of NUP98-HOXA10 on HSC
expansion was preserved when sequences flanking the homeodomain were
removed, thus identifying the homeodomain as the key HOX gene sequence
required in concert with the N-terminal region of NUP98.
These findings demonstrate a greater potency of NUP98-HOX fusions as
novel agents for HSC expansion ex vivo, reveal the essential contribution of the
DNA-binding homeodomain to achieve this effect and set the stage for the design
of minimal HOX-based fusion proteins for future studies.Medicine, Faculty ofMedical Genetics, Department ofGraduat
Characterization of mouse hematopoietic stem cells primed to actively self-renew by NUP98-HOXA10hd fusion gene
High-level expansion of hematopoietic stem cells (HSCs) in vitro will have an important clinical impact in addition to enabling elucidation of their regulation. Recently, it has been demonstrated that engineered NUP98-HOXA10hd expression stimulates >1,000-fold net expansions of murine HSCs in 10-day cultures initiated with bulk linâ»Sca-1âșc-kitâș cells. In this thesis I coupled such ability of engineered NUP98-HOXA10hd expression, with strategies to purify fetal and adult HSCs and analyze their expansion clonally. I discovered that NUP98-HOXA10hd stimulates comparable expansions of HSCs from both sources at near unit efficiency in cultures initiated with single cells. The clonally expanded HSCs showed preservation of normal proliferation kinetics in vitro and consistent balanced contributions long-term to the lymphoid and myeloid lineages in vivo without evidence of leukemogenic transformation. Preservation of a normal proliferating HSC phenotype allowed their re-isolation in large numbers at 25% purity. These findings point to the effects of NUP98-HOXA10hd on HSCs in vitro being mediated by promoting self-renewal and set the stage for further dissection of this process.
Although there is growing excitement about the prospect of in vitro expansion of HSCs and their use to enhance the safety and application of transplant-based therapies, deleterious consequences of such manipulations remain unknown. Thus, I further examined the impact of HSC self-renewal divisions in vitro and in vivo on their subsequent regenerative and continuing ability to sustain blood cell production in the absence of telomerase. HSC expansion in vitro was obtained using NUP98-HOXA10hd transduction strategy and, in vivo, using a serial transplant protocol. I observed ~10kb telomere loss in leukocytes produced in secondary mice transplanted with HSCs regenerated in primary recipients of NUP98-HOXA10hd-transduced and in vitro-expanded Tertâ»/â» HSCs 6 months before. The second generation leukocytes also showed elevated expression of ÎłH2AX (relative to control) indicative of greater accumulating DNA damage. In contrast, significant telomere shortening was not detected in leukocytes produced from freshly isolated, serially transplanted wild-type or Tertâ»/â» HSCs, suggesting that HSC replication post-transplant is not limited by telomere shortening in the mouse. These findings document a role of telomerase in telomere homeostasis, and in preserving HSC functional integrity upon prolonged self-renewal stimulation.Medicine, Faculty ofMedical Genetics, Department ofGraduat